**1. Introduction**

108 New Approaches to the Study of Marine Mammals

[71] Ministerio del Ambiente. Plan Nacional de Implementación para la Gestión de los Contaminantes Orgánicos Persistentes en el Ecuador," GEF/2732-02-4456. Global Environmental Facility (GEF), Convenio de Estocolmo sobreContaminantesOrgánicos

Persistentes (COPs), PNUMA, Ministerio delAmbiente del Ecuador; 2006.

The need to study, evaluate and understand the impacts of marine pollution on marine life is real and urgent. We depend on the ocean for food, transportation, economic gain, leisure and to enhance the quality of our lives. The residence times of pollutants in ocean water are short. The pollutants either settle to the bottom and attach to sediments or enter the food chain and accumulate in marine organisms. These outcomes mean that the only effective ways to assess marine pollution are to study the concentrations of pollutants in sediments or to study them in marine organisms. The average depth of the world ocean is 3,790 meters making it technically impractical to assess pollutants in sediments worldwide because of the great ocean depths under extreme pressures and vast amount of area. Thus, the best approach to assessing ocean pollution is to study it in marine organisms and, because of their relationship to humans (both biologically, culturally and inspirationally) and their ability to integrate air, water and prey, the best marine organisms to focus on are marine mammals.

There are many marine mammal species. Many of them are listed as endangered or threatened. A species that is considered to be one of the most endangered in the world is the North Atlantic right whale. Their population numbers only about 400 individuals [1]. This species suffers detrimental losses to their population through boat strikes and entanglements in fishing gear [1]. Regulations are being implemented to prevent extinction; however, boat strikes and entanglements may not be the only reason the population numbers remain so low. Other factors, perhaps pollutants, might be affecting the overall survival and reproductive ability of these animals [2].

© 2012 Wise et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ocean pollution is a threat to marine mammals. Some pollutants cause obvious and direct harm to the animals such as plastics and other debris. Others can be less evident, such as, agricultural runoff and industrial wastes. Industrial wastes can include air pollution that the animals breathe in and pollutants that can be found at different levels of the water column. These types of pollutants can have long term and persistent exposures.

Skin Biopsy Applications in Free Ranging Marine Mammals: A Case Study of Whale Skin Biopsies

developmental toxicant that is understudied in the marine environment [3]. Our approach is straightforward: 1) Determine if Cr exposure has occurred, 2) Determine if Cr is cytotoxic and genotoxic to cultured whale cells and 3) Compare data in cultured whale cells to levels in whale tissue to gain a toxic context. We exemplify this using biopsy samples from both

Whale skin biopsies were collected using a specialized biopsy dart and crossbow according to standard methods [4]. The biopsy dart has a stainless steel tip that collects a skin sample that is about 25 mm long or less and 7 mm in diameter. A buoyant stopper located behind the tip prevents the biopsy dart from penetrating beyond the depth of the tip. The stopper causes the dart to bounce off and float for an easy retrieval with a net. Tips are stored in 70% ethanol until use. Upon retrieval, the tissue sample is removed from the tip using Teflon

The skin and blubber were separated leaving the interface to be used for skin fibroblast cell growth. The blubber and skin were used for analysis of genetics, levels of metals and organics. The interface, once isolated, was immersed in a tissue buffer (PBS with 20% penn/strep and 2% gentamicin) for 30 minutes to get rid of any bacteria that may have been present on the skin. Tissue was then placed in a Petri dish and cut into approximately 1 mm pieces. These pieces were transferred into two T-25 flask with 1 ml of medium (DMEM-F12, cosmic calf serum, L-glutamine, penicillin, streptomycin, sodium pyruvate) and placed upside down in a 33°C humidified incubator with 5% CO2. After 24 h, 5 ml of medium was added and the flask was gently turned right side up and monitored for cell growth. Living cells typically plated out on the flask directly from the tissue explants within one week.

The whale skin biopsies were analyzed using inductively coupled plasma mass spectroscopy to determine the total chromium in the tissue according to published methods using a Perkin-Elmer/Sciex ELAN ICPMS. Samples were rinsed with deionized water and allowed to air dry in a laminar flow hood to minimize contamination. Approximately 0.1 g of tissue was placed in a digestion vessel, 2 ml of Optima grade nitric acid was added, the vessel placed in a hot block, and refluxed at 95°C for 4 h. The sample was cooled, 2 ml Optima grade hydrogen and deionized water (3:2 v/v) was added, heated until the effervescence subsided, cooled, and brought up to a final volume of 20 ml. Standard quality assurance procedures were employed (Table 1) and include the analysis of standard

sperm whales and North Atlantic right whales.

forceps and placed into a glass Petri dish.

*2.1.3. Measuring Levels in the Skin from the Biopsy* 

**2.1. Determining if Cr exposure has occurred in whales** 

**2. Materials and methods** 

*2.1.1. Biopsy collection* 

*2.1.2. Biopsy processing* 

as a Valuable and Essential Tool for Studying Marine Mammal Toxicology and Conservation 111

Whales are exposed to all environmental pollutants that reach the ocean. They spend time at the surface and travel throughout the water column so they are exposed to pollutants that remain at the surface and those that disperse through the water column. They experience dermal exposure through pollutants in the water. They feed at different depths so they get pollutants through ingesting animals that may have accumulated them. They all breathe air and, thus, are exposed to air pollutants. Consequently, they make excellent models to use for studying the threats and consequences of ocean pollution. The challenge, of course, is to develop an approach for studying the toxicology of marine pollution in marine mammals that provides species specific data along with an individual and population context.

Marine mammal research is difficult and expensive. There are laws that have been implemented to specifically protect marine mammals. Some species not only fall under this protection but also are protected by the Endangered Species Act. Thus, research with these animals is strictly enforced and regulated through various permitting agencies (eg, National Marine Fisheries Service (NMFS) and United States Fish and Wildlife Service, USFWS). These permits limit the amount, time and ways they can be studied. In addition to the permits, properly trained personnel and proper equipment are required. Whales in particular are difficult to study because they are typically found far from shore requiring extensive travel and vessel time. There is specialized equipment to aid in finding the whales while they are underwater but most of the search requires visual sightings of animals for the short period of time they are at the surface, requiring trained crew to be on watch during all daytime hours in a variety of environmental conditions. Because of these factors, researching whales is a particularly challenging task and one that requires careful use of resources and the ability to extract as much information as possible.

A unique and effective way to study marine mammals is through skin biopsies. The skin can be used to determine the genetics of an individual which can allow for gender identification as well as determining intraspecific relationships, genetic diversity of different subpopulations and tracking individual animals over time. Biopsies can provide important information about environmental pollutants and their effects. For example, they can be used to measure the levels of metals because many metals are known to accumulate in the skin. Through a skin biopsy, blubber is also collected which can be measured for a variety of organic pollutants. In addition, the interface between the skin and blubber can be used to create living cell lines. These cell lines can further be used to determine the toxicity of a pollutant by measuring levels of toxicity and DNA damage.

In this chapter we use chromium as an example for how skin biopsies of free ranging whales can be used to evaluate the environmental impact of a particular ocean contaminant. We chose to use chromium because it is a known carcinogen and a known reproductive and developmental toxicant that is understudied in the marine environment [3]. Our approach is straightforward: 1) Determine if Cr exposure has occurred, 2) Determine if Cr is cytotoxic and genotoxic to cultured whale cells and 3) Compare data in cultured whale cells to levels in whale tissue to gain a toxic context. We exemplify this using biopsy samples from both sperm whales and North Atlantic right whales.
